System, apparatus, and method for the removal of background stain by means of electric current from an electrophoresis supporting medium



Nov. 28, 1967 SYSTEM, APPARA'riJs BACKGROUND STAIN T G. FERRIS ETAL ANDMETHOD FOR THE REMOVAL BY MEANS OF ELECTRIC CURRENT FROM ANELECTROPHORESIS SUPPORTING MEDIUM Filed March 6, 1963 2 Sheets-Sheet lINVENTORS THOMAS G. FERRIS Nov. 28, 1967 T. G. FERRIS ETAL 3,355,372

SYSTEM, APPARATUS, AND METHOD FOR THE REMOVAL OF BACKGROUND STAIN BYMEANS OF ELECTRIC CURRENT FROM AN ELECTROPHORESIS SUPPORTING MEDIUMFiled March 6, 1963 2 Sheets-Sheet 2 INVENTORS THOMAS G. FERRIS ROBERTE. EASTERLING RICHARD E. BUDD by @n A 7' TO/PNEV United States PatentSYSTEM, APPARATUS, AND NETHOD FOR THE REMOVAL OF BACKGROUND STAIN BYMEANS OF ELECTRIC CURRENT FROM AN ELECTRO- PHORESIS SUPPORTING MEDIUMThomas G. Ferris, 5420 Alta Vista Road, Bethesda, Md. 20014; Richard E.Budd, 659 Wildwood Blvd., Williamsport, Pa. 17701; and Robert E.Easterling, 721 Courtland Drive {P.O. Box 711), Sanford, N.C. 27330Filed Mar. 6, 1963, Ser. No. 263,361 Claims. (Cl. 204-180) The inventiondescribed herein may be manufactured and used by or for the Governmentof the United States of America for governmental purposes without thepayment of any royalties thereon or therefor.

This invention relates to a system, apparatus, and method for theremoval of background stain by means of electric current from anelectrophoresis supporting medium.

A most common prior art method for removing background stain from anelectrophoresis supporting medium used for the fractionation, forexample, of serum proteins, has been by washing techniques, often bycirculating washing techniques. In certain cases, such as whenpolymerized acrylamide gel is the medium, the removal of the backgroundstain by washing is quite time-consuming, requiring perhaps from two tofive hours to complete. Arrangements for the removal of background stainby means of electric current have been published as indicated, forexample, in the article entitled, Electrophoresis of Serum Protein inAcrylamide Gel, by the present inventors, Ferris, Easterling, and Budd,appearing in the American Journal of Clinical Pathology, vol. 38, No. 4,pages 383- 387, October 1962, and ir1,the Instruction Bulletins for DiscElectrophoresis distributed by Canal Industrial Corporation, Bethesda,Md. However, these arrangements are in the one case limited to smallpieces of gel, and in both cases are apt to suffer from certaindeficiencies.

The deficiencies encountered with horizontal destaining apparatus, suchas that described in the aforementioned article of the inventors, aredue primarily to the evolution of gas at the electrodes. This gas issometimes trapped beneath the gel, interfering with the uniform andcomplete removal of the background stain. When the gel is in directcontact with the anode during destaining, bubbles form within the gel.These bubbles interfere with subsequent scanning of the gel in arecording densitometer. In horizontal destaining apparatus when the gelto be destained is smaller than the electrode plates, c.g., when the gelhas been sliced into strips containing individual electrophoreticpatterns prior to destaining, the current tends to shunt around the gelrather than passing through it. As a result, the destaining of the gelis inefiicient and often incomplete, even though destaining is carriedout beyond the thirty minutes which is routine with the presentinvention.

An object of the present invention, therefore, vide a system, apparatus,and method for the electrical destaining of an electrophoresissupporting medium which rapidly and efiectively removes the backgroundstain and leaves clearly defined electrophoretic patterns with anadequately clear background in the electrophoresis medium to facilitateaccurate scanning and evaluation of the electrophoretic patterns.

Other objects, advantages and novel features of the invention willbecome apparent from the following detailed description when consideredin conjunction with the accompanying drawings wherein:

FIG. 1 is an exploded elevation view of a preferred embodiment of theapparatus of the invention;

is to pro- FIG. 2 is an axonometric, generally top view of a slab of gelin generally horizontal position adhered to a piece of filter paper;

FIG. 3 is a vertical cross section taken along the line 3-3 in FIG. 4 ofthe apparatus of FIG. 1 assembled and with the gel-filter paper laminateof FIG. 2 in position for destaining; and

FIG. 4 is a cross section taken along the line 4-4 in FIG. 3.

Reference is now made to the drawings. The numeral 2 indicates acontainer which for convenience can be an ordinary cylindrical batteryjar. Lid 4, conveniently of clear plastic, is provided to insure goodvisibility of the contents of the container and to protect against thedanger of accidental electric shock. The lid is provided with holes 6through which can be inserted insulated connectors 8 and 10 forconnecting to a source of electricity the prongs 12 and 14 of the anode16 and cathode 18, respectively. The cylindrical electrodes 16, 18 areheld in fixed, generally concentric relation to each other by anyconvenient means, such as a press fit, serving to fasten them to aninsulating base 20. Typically, the anode 16 may be tightly sweated ontothe base 20, readily made of plastic, with such a good fit that liquidsuch as the electrolyte 22 is contained in the annular space between thetwo electrodes, the container 2 then serving merely as a precautionagainst leakage.

In FIG. 2 there is shown a slab 24 of electrophoresis supporting medium,such as polymerized acrylamide gel, adhered to a sheet of filter paper26 to form a laminate. In FIGS. 1, 3 and 4 the lam nate is shown ashaving been rolled into a cylinder with the slab of gel on its interiorand the filter paper being fastened at its overlapping ends by suchconvenient means as staples 28. Bafile 30 of insulating material isshown transversely spanning those lines of the electric field betweenthe two electrodes which do not intersect the electrophoresis supportingmedium.

To facilitate an understanding of the system apparatus and method of theinvention, a description of the invention is given herewith as applied,by way of example only, to the destaining of a particular gel used inserum protein electrophoresis. To provide an informative backgroundsetting to elucidate the use of the invention, a brief description isgiven of the preparation of the electrophoresis supporting medium.

Serum protein electrophoresis is carried out in a verticalelectrophoresis cell in the manner mentioned in the previouslyreferenced article in the American Journal of Clinical Pathology using apolymerized acrylamide gel in the manner analogous to that used forhemoglobin electrophoresis described in the article by Ferris,Easterling, and Budd, appearing in Blood Volume 19, No. 4, April 1962,pages 479-481. Although other supporting media for zone electrophoresiscan be used, the one chosen for this example is the acrylamide gel knownas Cyanogum 41 described in the article by Raymond and Weintraubpublished in Science, Sept. 18, 1959, page 711, and in the bulletinpublished by the American Cyanamid Company entitled, Cyanogum 41Gel-ling Agent for Industrial Use. After electrophoresis is completed,the gel is carefully removed from the cell and immediately placed in astaining solution. The gel may typically be 5" x 7" x mm. thick. Variousstains can be used, but an excellent stain for serum proteins is thestain known as Amidoschwartz 10B, also known as Buffalo Black NBR,Naphthol Blue Black and Acid Black I, appearing in the Color Index underCI20470. A stock stain solution can be made of one gram of Amidoschwartz10B dissolved in ml. methanol to which is added 700 ml. glacial aceticacid and qs to one liter with distilled water. A working stain solutionis made by diluting the stock solution 1:1 with distilled water justprior to use.

T he protein is fixed in the gel and stained simultane- "ous by thisprocedure. It is necessary to leave the gel in the stain long enough tocompletely fix and stain the lproteins. If the gel is removedprematurely, part of the Eprotein will not be fixed and will be removedfrom the :eel along the background stain, during the destaininglproce'ss. For convenience, the gel can be left in the work- :in'g stainovernight (16 hours). Staining and fixing can e carried out in 2 hoursif the gel is placed in the stock stain solution instead of the workingstain solution.

After the staining-fixing is completed, the gel is carefully removedfrom the stain solution and laid out on a fiat, smooth surface, such asa piece of glass. A piece of heavy dry filter paper, preferably somewhatlarger than the gel is gently pressed onto the gel as shown in FIG. 2.The gel and filter paper will adhere and will remain adhered even whenthe paper is held in a vertical position. The filter paper used is aheavy filter paper which remains rigid when wet. The filter paper-gellaminate is formed into a cylinder with the gel on the inside of thepaper cylinder. The laminate is retained in the cylinder form by anyconvenient means such as by overlapping the ends of the filter paper andstapling the filter paper together in several places along the seam asshown in FIGS. 1, 3 and 4. The cylinder laminate is then carefullyplaced in the destaining apparatus and takes the position shown in F165.3 and 4. To proceed with the setup of the system of the invention, theapparatus is filled with sufiicient 115% acetic acid solution to bringthe electrolyte level just over the upper edge of the gel.The'concentration is not critical, but best results are obtained withacetic acid solutions in therange of 1G%l5%. Other electrolytes can beused, but acetic acid is a logical choice since acetic acid is used tofix the protein during the staining-fixing part of the procedure and itsmolecule is small permitting rapid penetration of the gel.

The paper-gel laminate cylinder is adjusted so that it is approximatelyconcentric with the apparatus and the insulating baffle shown in FIGS.1, 3 and 4 is placed to prevent the shunting of current through the areabetween the ends of the gel, permitting more efficient destaining of thegel. The insulating bafile is not absolutely essential for destaining,but its use appreciably improves efiiciency. If the ends of the gelareapproximated, so that the cylinder of gel is'essentially continuousand without a gap such as seen in FIG. 4, then the bafiie isunnecessary. Such a configuration can be achieved by, for example,placing the filter paper with one ultimately vertical edge flush with anultimately vertical edge of the gel instead of the two opposite edges ofthe paper overhanging the gel, as in the illustrated embodiment. Thelaminate cylinder would then be formed by rolling the laminate into aposition with the gel ends abutting and with the single projecting endof filter paper overlapping,

on the outside of the cylinder, its opposite end which is flush with thegel surface. The overlapping portions of filter paper would then befastened together by any suitable fastening mean s, such as adhesive ortape, which remain fastened when wet outdo not hinder the electrical.destaining process.

The lid 4 is now placed on the container 2, and the leads 3 and 10 fromthe direct current power supply :are attached to the prongs 12 and 14 sothat the outer electrode 16 is the anode, Various combinations of timeand current can be used for destaining. With a gel slab of theaforementioned size, a constant current of one :ampere applied forthirty minutes has been found to produce excellent results. The currentapplied is reduced in proportion to the reduction in gel area. Forexample, if a piece of gel to be destained is half the Width of theaforementioned uncut gel, then the current applied would .be 0.5 ampereinstead of 1 ampere used to destain the 4 whole gel slab. *If a slab ofgel, alonewithout filter aper backing, is formed into a cylinder thecurvature of the gel gives it sufficient rigidity to stand upright inthe electrolyte. However, under such circumstances, the gel tends tocling to theouterelectrode and although it can be satisfactorilydestained by this procedure, bubbles form within the gel when the gel isin direct contact with the anode during the destaining. These bubblesinterfere, as previously noted, with subsequent scanning of the gel in arecording densitometer. In addition, the gel tends to be slippery andhard'to handle, and it is sometimes difficult to get the gel into theapparatus without tearing it. The filter paper cylinder technique solvesboth the problem of gel support and gel positioning between the twoelectrodes. The paper cylinder also provides a convenient means ofgetting the gel into and out of the apparatus without damage to the gel.It has the additional advantage of acting as a barrier to thecirculation of removed stain throughout the electrolyte. That is, withthe'electric current passing'between the two electrodes, the stain ispulled out of the gel and toward the anode 16 by the electric current;and as the stain passes through the paper cylinder and into theelectrolyte, it is trappedin the annular space between the wall of theanode and the paper..

As the destaining progresses, the electrolyte between the paper and theanode becomes very dark with removed stain while theelectrolytebetweenthe paper-gel laminate and the cathode remainsrelatively clear. The light coming through the transparent bottom of theapparatus makes the gel readily visible. As destaining proceeds, the gelcan be seen to become transparent as the background stain is removed bythe electric current.

After destaining is completed, the current is shut off and the leads 8and 10 disconnected. The paper-gel laminate cylinder is carefullyremoved from the apparatus and placed in a shallow pan of water. Thepaper is cut apart along the stapled seam and the laminate is. permittedto lie flat in the water, gel side down. The filter paper can now beeasily removed by gently pulling the paper away from the gel while thegel remains submerged in the water. I v

Although the invention has been illustrated as using acrylamide gel, itis also applicable to other electrophoresis "supporting media such, forexample, as starchgel.

Obviously, many modifications and variations of the present inventionare possible in the light of'the above teachings. It is therefore to beunderstood.'that within the scope of the appended claims the inventionmay be practiced otherwise than as specifically described.

What is claimed is: f

1. A method of removing stain from an electrophoresis supporting mediumcomprised of 'a slab of gel which includes: l

adhering said slab of gel to filter'paper,

configuring the laminate formed of said gel and, filter paper into agenerally cylindrical shape with the gel on the interior thereof, H vfastening the adjacent end portions of the filter paper together toretain the. laminate in its generally cylindrical,relatively rigid form,disposing said laminate generally vertically concen-v trically inelectrolyte between a pair of generally concentrically spaced generallycylindrical electrodes and free of contact with each electrode,maintaining the electrolyte at a level essentially just covering theupper edge of said gel, and

applying a direct electric potential difference between said electrodesto attract stain from said gel through said filter paper toward theouter electrode.

2. The method of claim 1 wherein said gel comprises polymerizedacrylamide. v

3. The method of claim 1 further including the step of interposingduring the application of electric potential difference, bafiic meansbetween said electrodes spanning transversely those radii of the radialelectric path through the electrolyte between said electrodes which donot intersect said electrophoresis, supporting medium.

4. A method of removing stain from an electrophoresis supporting mediumcomprised of a slab of gel which includes:

adhering said slab of laminate;

disposing said laminate generally vertically in electrolyte between, butfree of contact with, a pair of electrodes, the surfaces of theelectrodes being generally parallel to the gel-paper interface of saidlaminate; and

applying a direct electric potential ditterence between said electrodesto attract stain from said gel toward one of said electrodes.

5. The method of claim 4 wherein the gel is polymerized acrylamide.

gel to filter paper to form a References Cited UNITED STATES PATENTS1,235,064 7/ 1917 Schwerin 204-480 1,266,329 6/1918 Schwerin 204180 61,815,302 7/1931 Hogstad 204-480 1,878,235 9/1932 Gortner et a1. 2041802,741,595 4/1956 Juda 204301 OTHER REFERENCES Barka: The Journal ofHistochemistry and Cytochernistry, Studies of Acid Phosphatase, I.Electrophoretic Separation of Acid Phosphatase of Rat Liver onPoiyacrylamide Gels, vol. 9, No. 5, September 1961, pp. 542547.

Smithies: J. Biochemistry, An Improved Procedure for Starch GelElectrophoreses; Further Variations in the Serum Proteins of NormalIndividuals, vol. 71, 1959, pp. 585587.

Heftman: Chromatography, pp. 262-265, 1961.

Raymond: Analytical Biochemistry, Preparation and Properties ofAcrylarnide Gel for use in Electrophoresis, pp. 391-396, vol. 1, 1960.

JOHN H. MACK, Primary Examiner. HOWARD S. WILLIAMS, Examiner. J.BATTIST, E. ZAGARELLA, Assistant Examiners.

1. A METHOD OF REMOVING STAIN FROM AN ELECTROPHORESIS SUPPORTING MEDIUM COMPRISED OF A SLAB OF GEL WHICH INCLUDES: ADHERING SAID SLAB OF GEL TO FILTER PAPER, CONFIGURING THE LAMINATE FORMED OF SAID GEL AND FILTER PAPER INTO A GENERALLY CYLINDRICAL SHPAE WITH THE GEL ON THE INTERIOR THEREOF, FASTENING THE ADJACENT END PORTIONS OF THE FILTER PAPER TOGETHER TO RETAIN THE LAMINATE GENERLLY VERTICALLY CONCENTRICALLY IN ELECTROLYTE BETWEEN A PAIR OF GENERALLY CONCENTRICALLY SPACED GENERALLY CYLINDRICAL ELETRODES AND FREE OF CONTACT WITH EACH ELECTRODE, MAINTAINING THE LEECTROLYTE AT A LEVEL ESSENTIALLY JUST COVERING THE UPPER EDGE OF SAID GEL, AND APPLYING A DIRECT ELETRIC POTENTIAL DIFFERENCE BETWEEN SAID ELECTRODES DO ATTRACT STAIN FROM SAID GEL THROUGH SAID FILTER PAPER TOWARD THE OUTER ELECTRODE. 